Current methods to isolate/identify live human embryonic and induced pluripotent stem cells or to monitor the process of reprogramming somatic cells, use antibodies against surface markers e.g. SSEA-4, SSEA-3, Tra-160, Tra-181 or have the cells genetically modified to report the expression of pluripotency genes, such as Oct-4 or Sox-2. Use of antibodies is subject to variability and/or genetic modification has obvious inherent safety issues if these cells are to be used in therapy later on. Other methods have used colony morphology or nuclear to cytoplasmic ratio, though this method is not definitive i.e. is only suggestive. For e.g. colony morphology does not address single cell isolation and has a strong subjective component. Conversely these methods are used to isolate differentiated cells away from teratoma-initiating pluripotent cells. A major thrust of human ES and iPS cells will be to generate specific differentiated cell types from pluripotent cells. Since these conversions are rarely complete, it would be important to eliminate the pluripotent cells within the differentiated population of cells. The pluripotent cells, if present, can differentiate to either to cells that are not desired or to tumor forming cells i.e. teratomas, which can interfere in the therapeutic use and in addition increase the possibility of malignant and benign tumors. The endogenous blue fluorescence can be used to isolate undifferentiated pluripotent cells from the differentiated cells.
A recent report had used fluorescence life-time measurements (FLIM) of endogenous fluorophores to discriminate between pluripotent and differentiating human embryonic stem cells using a custom-designed multiphoton microscope, fluorescence lifetime measurements along with phasor analysis. They reported that the fluorescence in the blue region emanating from pluripotent cells arose from two entities—NADH and LDAG (lipid droplet associated granules). LDAG are aggregates of lipid bodies, which are a mixture of neutral lipids contained by a monolayer of phospholipids and may also be associated with some proteins. The composition of lipids and proteins present in these bodies can vary within the cells and also between cells. They also state that the ratio of the levels of NADH fluorescence to the fluorescence emanating from lipid aggregates termed) within these cells are used to identify pluripotent human embryonic stem cells. The document used a multiphoton excitation source to measure the fluorescence lifetimes, subjected the intensities measured to phasor analysis to determine individual fluorescence lifetimes. These were stated to be plotted as a phasor plot to identify the sources and characteristics of the emitted fluorescence and estimate the differentiation of the pluripotent cell population. The method used a custom-designed FLIM microscope along with phasor analysis. The paper also emphasizes in multiple places that the relative ratio of the LDAG fluorescence to NADH fluorescence is used to determine the undifferentiated status. The report also did not demonstrate isolation/separation of pluripotent human cells from their differentiating counterparts. The above method has also not been applied to human induced pluripotent stem cells, mouse embryonic stem cells or mouse epiblast stem cells (mEpiSCs). The document also states that the fluorescence seen in the LDAG emanated from the reaction of lipid peroxides with proteins. The lipid peroxides are multiple chemical entities which are generated by the oxidation of lipids within the lipid bodies by reactive oxygen species (ROS), depending on the type of lipid molecules that are present. In particular, lipids that are unsaturated are more prone to oxidation. The document (Chiari et al.) did not identify or isolate any specific lipid or its oxidation product but speculated this to be so. It also stated that human ES cells have high ROS values compared to other cells and hence these bodies (LDAG) are fluorescent in human embryonic stem cells. However, there are various other literatures which indicate that ROS values in embryonic stem cells are lower than in differentiated cells.
The prior art document as aforementioned used a custom-designed FLIM microscope, not easily available, is expensive and technically challenging (for e.g. requires a femtosecond laser for excitation and requires measurement and separation of individual fluorescence lifetimes) and does not lend to FACS sorting. This technique is not suited for high throughput and cannot be compared favourably to sorting by FACS. The report also did not demonstrate physical isolation of pluripotent stem cells and subsequent culture of these cells or examined induced pluripotent stem cells or epiblast-like stem cells. The images provided by the FLIM microscope do not lend to easy mechanical dissection due to their low resolution. The method also depends on the fluorescence lifetimes of the fluorophores which are affected by the molecular environment in which the fluorophores reside and can show substantial variation and is highly context-dependent. For e.g., a master's thesis from the same laboratory had previously reported FLIM analysis of human embryonic stem cells and given a different interpretation/identification to the fluorescence observed. The paper also does not report the absence of lipid bodies and associated fluorescence in mouse ES cells, does not report any experiments with human induced pluripotent cells, and also does not associate the endogenous blue fluorescent lipid bodies with the epiblast-like stem cell state.
The instant disclosure overcomes the drawbacks in the prior art by describing a method which measures just the intensity of the endogenous blue fluorescence (i.e. autofluorescence) using standard wide-field epifluorescence microscopes to identify pluripotent stem cells. In addition it shows that conventional fluorescence activated cell sorting (FACS) is used to isolate pluripotent cells from differentiating cells again based on just the intensity of blue fluorescence. In other words, it does not require the measurement of fluorescence lifetimes and their analysis or the need to measure the ratio of LDAG fluorescence and NADH fluorescence to identify and isolate pluripotent cells. The instant disclosure also therefore easily lends itself to high throughput identification and isolation, for e.g. FACS, and subsequent propagation of human pluripotent stem cells unlike the FLIM method. The instant method also further demonstrates its application in isolating single human pluripotent stem cells (i.e. FACS is dependent on dispersion into single cells) and subsequent propagation, which has been a serious limitation in the prior art. This method also avoids the variability associated with antibody labeling or genetic modification.
The method described in the instant disclosure, in addition to examining the above, is robust can be easily and directly applied with standard equipment available in most laboratories and does not require specific expertise or training or very sophisticated instruments. Hence, the instant disclosure overcomes all the drawbacks presently being faced in the prior art and improves the current field of technology.